Methods for processing biological samples by means of laser microdissection have existed since the mid-1970s (see e.g. Isenberg, G. et al.: Cell surgery by laser microdissection: a preparative method, Journal of Microscopy, volume 107, 1976, pages 19-24) and have since been continuously developed.
In laser microdissection, cells, tissue regions, etc. can be isolated from an object (e.g. a “sample,” or a “preparation”) and extracted as dissection specimens. A particular advantage of laser microdissection is the brief contact between the object and the laser beam, as a result of which said object is barely modified. The specific extraction of the dissection specimens can be carried out in various ways (see e.g. Bancroft, J. D. and Gamble, M.: Theory and Practice of Histological Techniques, Elsevier Science, 2008, page 575, “Laser Microdissection” chapter).
For example, in known methods, a dissection specimen can be isolated from an object by means of an infrared laser beam or ultraviolet laser beam, which specimen falls into a suitable dissection specimen collection container under the influence of gravity. The dissection specimen can also be cut out of the object in this case, together with an adherent membrane. In laser capture microdissection, however, a thermoplastic membrane is heated using a corresponding laser beam. In this case, the membrane fuses with the desired region of the object and can be removed by means of tearing in a subsequent step. A further alternative is that of attaching the dissection specimen to a lid of a dissection specimen collection container by means of the laser beam. In known inverse microscope systems for laser microdissection, dissection specimens which are ejected upwards can also be attached to the base of a dissection specimen collection container which is provided with an adhesive coating.
Known microscope systems for laser microdissection, as are known from WO 98/14816 A1 for example, comprise a reflected light device, into the beam path of which a laser beam is coupled. The laser beam is focussed on the object through the microscope objective used in each case, which object is rests on a microscope stage which can be automatically moved by means of a motor. A cutting line is produced by the microscope stage being moved during cutting, in order to move the object relative to the stationary laser beam. However this has the disadvantage, inter alia, that it is not easy to observe the object while the cutting line is being produced, since said object moves in the field of vision and the image thereof may appear blurred.
Laser microdissection systems which have laser deflection devices and laser scanning devices designed to direct the laser beam or the point of impingement of said beam over a stationary object are therefore more advantageous. Laser microdissection systems of this kind, which also provide particular advantages within the scope of the present invention, are explained in detail hereinafter. A particularly advantageous laser microdissection system having a laser deflection device, which comprises glass wedges in the laser beam path which can be adjusted relative to one another, is described in EP 1 276 586 B1 for example.
In both cases, i.e. in both laser microdissection systems in which the microscope stage is moved and laser microdissection systems comprising a laser deflection device, pulsed lasers are generally used, each laser pulse creating a hole or depression in the object. A cutting line is produced as a result of a series of successive, and possibly overlapping, holes or depressions of this kind. Laser microdissection can be used to extract individual cells or defined tissue regions which then undergo different diagnostic analysis methods, for example. In oncology, laser microdissection can be used, for example, to isolate specific tumour cells from a microscopic cut and examine them for specific metabolites or proteins. In this case, it has to be ensured that no material or as little material as possible from undesired and possibly interfering regions of the examined object reaches an examination vessel and therefore interferes with a corresponding analysis. This also applies to laser microdissection systems which are used for molecular biological examinations, for example for known COMET assays.
The accuracy of the laser used is therefore crucial for successful and uncontaminated laser microdissection experiments. The more accurately the laser is able to cut, for example with the aid of predefined cutting lines, or can be guided over the region in question with precise accuracy, the cleaner the extraction of corresponding material.
The laser of a laser microdissection system or a corresponding laser deflection device therefore has to be calibrated as accurately as possible, so that default position values, which indicate target positions for the points at which the laser impinges on the sample, and the resulting actual position values, i.e. the actual points of impingement, differ from one another as little as possible.
Common calibration methods are, however, often not sufficiently reliable or exact and/or are very complex for a user to carry out.